Synthesis and antimetastatic effects of E-salignone amide derivatives

Article abstract of DOI:10.1002/ddr.21157

Preclinical Research The preparation of novel E-salignone derivatives and their biological evaluation as potential antimetastatic agents is described. The E-salignone amide derivatives were prepared from epiandrosterone and androsterone, and characterized by analytical ¹H NMR, ¹³C NMR, and mass spectrometry. The derivatives were evaluated for antimetastatic activity in MDA-MB-231 cells by using a transwell assay. Comparing with the positive control, LY294002, compounds 19b, 19d, and 19e exhibited significant inhibitory effects on the EGF-induced invasion of MB-MDA-231 cells. Moreover, compound 19b also had antimigration effects in wound-healing assay. Compound 19b may represent a novel antimetastatic agent for treating breast cancer.

Full text of DOI:10.1002/ddr.21157

DRUG DEVELOPMENT RESEARCH 75 : 76–87 (2014)
Research Article
Synthesis and Antimetastatic Effects of E-Salignone
Amide Derivatives
Hong-Ling Wang,1† Nan Qin,2† Jia Liu,3 Mei-Na Jin,3 Xiang Zhang,1 Mei-Hua Jin,3
Dexin Kong,2,3 Shen-De Jiang1, and Hong-Quan Duan2,3*
1School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
2Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China
3Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics
and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University,
Tianjin 300070, China
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Technology,
Genomics,
Proteomics
Preclinical
Research
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics
Phases I-III
Regulatory, Quality,
Manufacturing
Postmarketing
Phase IV
ABSTRACT
The preparation of novel E-salignone derivatives and their biological evaluation as potential
antimetastatic agents is described. The E-salignone amide derivatives were prepared from epiandrosterone
and androsterone, and characterized by analytical 1H NMR, 13C NMR, and mass spectrometry. The
derivatives were evaluated for antimetastatic activity in MDA-MB-231 cells by using a transwell assay.
Comparing with the positive control, LY294002, compounds 19b, 19d, and 19e exhibited significant
inhibitory effects on the EGF-induced invasion of MB-MDA-231 cells. Moreover, compound 19b also had
antimigration effects in wound-healing assay. Compound 19b may represent a novel antimetastatic agent for
treating breast cancer. Drug Dev Res 75 : 76–87, 2014.
© 2013 Wiley Periodicals, Inc.
Key words: E-salignone; amide derivatives; antimetastasis; breast cancer cells
INTRODUCTION
basement membrane and extracellular matrix constitu-
ents; the ability to enter the bloodstream or lymphatic
system, and then survive in the circulation; and the
consequent establishment of new tumors [Mego et al.,
2010; Yachida et al., 2010].
Natural products (NPs) are a rich source of new
drugs. E-Salignone, an NP isolated from Pachysandra
terminalis, has inhibitory effects on the migration of
human MDA-MB-231 breast cancer cells induced
Metastasis, the dissemination and growth of neo-
plastic cells in an organ distinct from that in which they
originated, is a major cause of mortality in millions of
cancer patients, especially in breast cancer, the leading
cause of cancer-related death in women worldwide
[Jemal et al., 2011]. Preventing or inhibiting breast
cancer metastasis is key to prolonging and/or enhancing
the quality of life of patients. Treatment options for
metastatic breast cancer often focus on relieving symp-
toms together with systemic therapies to control
primary tumor growth, e.g., chemotherapy and hor-
†These authors contributed equally to this work.
*Correspondence to: Hong-Quan Duan, Tianjin Key
Laboratory on Technologies Enabling Development of Clinical
monal therapy. Unfortunately, only limited progress has Therapeutics and Diagnostics (Theranostics), School of Pharmacy,
been made in developing antimetastatic agents [Chen
Tianjin Medical University, Tianjin 300070, China.
E-mail: duanhq@tijmu.edu.cn
et al., 2010].
Received 29 August 2013; Accepted 8 October 2013
Cell migration plays a central role in metastasis
and involves multiple steps, including: loss of cell–cell
adhesion capacity; the degradation and invasion of .com). DOI: 10.1002/ddr.21157
Published online in Wiley Online Library (wileyonlinelibrary
© 2013 Wiley Periodicals, Inc..

ANTIMETASTATIC E-SALIGNONE AMIDE DERIVATIVES
77
Fig. 1. The synthesis of E-salignone and its amide derivatives.
by the chemokine epidermal growth factor (EGF-
IC50 = 0.87 μM) [Zhai et al., 2012]. As a result, the syn-
thesis of a series of novel E-salignone amide derivatives
was undertaken that were tested for their antitumor
metastatic activity.
Synthetic Procedures
Wittig olefination of epiandrosterone and and-
rosterone as starting material gave intermediates 1 and 2
stereoselectively [Deng et al., 1999] with the 3-OH of 1
and 2 being converted to phthalimide group by
Mitsunobu reaction (Fig. 1). This method of inversion of
the stereochemistry from the 3α-ol of 1 represented a
substantial advantage over other literature methods as
Materials and Methods
All solvents and reagents were obtained from the 3β-amine was isolated as a single diastereoisomer in
commercial sources and used without purification. good yield without the need for chromatographic puri-
Reactions were monitored by thin-layer chromatogra- fication of the intermediates [Hamilton et al., 2012]. The
phy (TLC) using precoated silica gel aluminum plates intermediates 3 and 4 were oxidized by Riley reaction to
containing a fluorescence indicator. The column chro- provide 16α-ol of 9 and 10 [Bruttomesso et al., 1999].
matography was carried out on silica gel (300–400 After the protection of the 3-NH2 group by di-tert-butyl
mesh) (Qingdao Haiyang Chemical Co. Ltd., Qingdao, dicarbonate, the 16α-ol of 13g and 14g were oxidized by
Shandong province, China). 1H, 13C NMR spectra were activity MnO2 to obtain the derivatives 15 and 16. After
taken using a Brüker AVANCE III 400 instrument the deprotection by TFA, the C-3 amino intermediates
(Bruker Biospin AG, Fallanden, Switzerland). 1H NMR 17 and 18 were obtained. Finally, all the amide deriva-
spectra were recorded in CDCl3 and DMSO-d6 using tives were prepared by condensation of the acids with
tetramethylsilane (TMS) as internal standard. Mass 1,1’-Carbonyldiimid-azole (CDI) or the acid chlorides
spectra were taken in ESI mode on an Agilent 1200 with 4-Dimethylaminopyridine (DMAP). The che-
LC-MS (Agilent, Palo Alto, CA, USA).
mical structures of all target compounds (Fig. 2) were
Drug Dev. Res.

78
WANG ET AL.
Fig. 2. The structures of E-salignone and its amide derivatives.
1
characterized by H NMR, 13C NMR, and ESI-MS (17Z )-Pregn-17(20)-en-3β-ol (1)
spectral data analyses.
Yield: 93.8%. 1H NMR (400 MHz, CDCl3): δ 5.11
(q, J = 6.9 Hz, 1H), 3.59 (m, 1H), 1.65 (d, J = 6.9 Hz,
3H), 0.87 (s, 3H), 0.82 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 150.5, 113.2, 71.3, 56.3, 54.4, 44.8, 44.4, 38.2,
37.2, 37.0, 35.5, 31.9, 31.4, 28.7, 24.4, 21.4, 16.9.
ESI-MS m/z: 301 [M-H]−.
General procedure of compounds 1 and 2
A mixture of t-BuOK (4.0 eq) and Ph3PEtBr
(4.0 eq) was dissolved in anhydrous tetrahydrofuran
(THF), stirred at room temperature for 1 h, then was
added the starting material androsterone or epian- (17Z )-Pregn-17(20)-en-3α-ol (2)
drosterone (1.0 eq). The mixture was refluxed for 4 h,
Yield: 91.2%. 1H NMR (400 MHz, CDCl3): δ 5.12
and saturated NH4Cl aqueous solution was added to (qt, J1 = 2.0, J2 = 7.1 Hz, 1H), 4.06 (s, 1H), 1.67 (dt,
quench the reaction. The reaction mixture was extracted J1 = 2.0, J2 = 7.1 Hz, 3H), 0.88(s,3H), 0.81(s,3H). 13C
with dichloromethane, washed with water, and dried NMR (100 MHz, CDCl3): δ 150.5, 113.2, 66.6, 56.3,
over MgSO4 and concentrated under reduced pressure 54.3, 44.4, 39.1, 37.2, 36.2, 35.9, 35.0, 32.2, 31.9, 31.4,
to obtain the crude product, which was purified by 29.0, 28.5, 24.3, 21.0, 16.9, 13.1, 11.2. ESI-MS m/z: 301
recrystallization with methanol to give white needles.
[M-H]−.
Drug Dev. Res.

ANTIMETASTATIC E-SALIGNONE AMIDE DERIVATIVES
79
General procedure of compounds 3 and 4
J1 = 2.0, J2 = 6.8 Hz, 3H), 0.86 (s, 3H), 0.79 (s, 3H). 13C
NMR (100 MHz, CDCl3): δ 150.5, 113.2, 56.3, 54.5,
51.1, 45.5, 44.4, 37.6, 37.2, 35.6, 35.1, 31.9, 31.4, 31.3,
28.7, 28.6, 24.4, 21.3, 16.9, 13.1, 12.3. ESI-MS m/z: 300
[M-H]−.
Ph3P (1.1 eq), Phathalimide (1.1 eq), and Com-
pound 1 or 2 (1.0 eq) were added to anhydrous THF.
The mixture was stirred in ice bath for 1 h, then
Diisopropyl azodicarboxylate (DIAD) (1.1 eq) was
added, stirred continuously hermetically for 18 h, and
distilled water was added to quench the reaction. THF
was evaporated to dryness. The reaction mixture was
extracted with dichloromethane, washed with water,
and dried over MgSO4. The solvent was evaporated
under reduced pressure to obtain the crude product,
which was purified by recrystallization with methanol
and dichloromethane to give white solid.
(17Z )-Pregn-17(20)-en-3α-amine (6)
Yield: 89.2%. 1H NMR (400 MHz, CDCl3): δ 5.10
(q, J = 6.8 Hz, 1H), 3.16 (br s, 1H), 1.64 (d, J = 6.8 Hz,
3H), 0.86 (s, 3H), 0.79 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 150.2, 113.1, 56.2, 54.4, 49.4, 45.6, 44.3, 39.1,
37.1, 36.3, 34.9, 32.0, 31.8, 31.3, 28.9, 28.6, 24.3, 20.9,
16.8, 13.0, 11.2. ESI-MS m/z: 300 [M-H]−.
(17Z )-3β-(1,3-Dioxoisoindolin-2-yl)-pregn-
17(20)-en (3)
(17E )-3β-Aminopregn-17(20)-en-16α-ol (11)
1
Yield: 88.1% H NMR (400 MHz, CDCl3) δ 5.57
1
Yield: 66.3%. H NMR (400 MHz, CDCl3): δ
(q, J = 6.8 Hz, 1H), 4.42 (d, J = 4.4 Hz, 1H), 2.66 (br s,
1H), 1.73 (d, J = 6.8 Hz, 3H), 0.86 (s, 3H), 0.80 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 155.6, 119.3, 74.4 54.5,
52.5, 51.1, 45.5, 44.5, 39.4, 37.6, 37.4, 35.6, 35.1, 34.4,
32.6, 31.9, 28.7, 21.2, 17.6, 13.2, 12.3. ESI-MS m/z: 316
[M-H]−
7.82–7.79 (m, 2H), 7.70–7.68 (m, 2H), 5.13 (qt, J1 = 1.9,
J2 = 7.2 Hz, 1H), 4.18 (m, 1H), 1.66 (dt, J1 = 1.9,
J2 = 7.2 Hz, 3H), 0.97 (s, 3H), 0.88 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 168.5, 150.5, 133.8, 132.1, 123.0,
113.2, 56.2, 54.1, 50.6, 46.1, 44.4, 37.8, 37.2, 35.6, 35.1,
31.8, 31.7, 31.4, 28.6, 25.1, 24.4, 21.3, 16.9, 13.1, 12.2.
ESI-MS m/z: 430 [M-H]−.
(17E )-3α-Aminopregn-17(20)-en-16α-ol (12)
Yield: 89.4%. 1H NMR (400 MHz, CDCl3): δ 5.58
(q, J = 7.2 Hz, 1H), 4.43 (d, J = 4.9 Hz, 1H), 3.21 (br s,
1H), 1.74 (dt, J = 7.2 Hz, 3H), 0.87 (s, 3H), 0.81 (s, 3H).
13C NMR (100 MHz, CDCl3): δ 155.5, 119.3, 74.3, 54.4,
52.5, 45.8, 44.6, 39.1, 37.4, 36.4, 36.0, 35.1, 32.0, 31.8,
28.9, 28.6, 20.8, 17.6, 13.2, 11.3. ESI-MS m/z: 316
[M-H]−.
(17Z )-3α-(1,3-Dioxoisoindolin-2-yl)-pregn-
17(20)-en (4)
1
Yield: 61.5%. H NMR (400 MHz, CDCl3): δ
7.81–7.82 (m, 2H), 7.71–7.69 (m, 2H), 5.12 (q,
J = 6.8 Hz, 1H), 4.51 (br s, 1H), 1.66 (d, J = 6.8 Hz,
3H), 0.89 (s, 3H), 0.87 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 169.2, 155.6, 133.8, 132.1, 122.9, 119.3, 74.4,
54.5, 52.4, 47.5, 44.5, 40.4, 37.4, 35.1, 35.0, 34.4, 32.7,
31.6, 31.6, 28.5, 24.6, 20.9, 17.6, 13.2, 12.4. ESI-MS
m/z: 430 [M-H]−.
General procedure for compounds 9 and 10
SeO2 (1.0 eq) and t-BuOOH (1.6 eq) were dis-
solved in CH2Cl2 solution, stirred in ice bath for 1 h
(Fig. 1). Compound 3 or 4 (1.0 eq) was dissolved in
CH2Cl2 solution, then was added into the mixture, the
reaction mixture was stirred continuously in ice bath for
3.5 h, 10% NaHSO3 was added then stirred for 15 min
to quench the reaction. The reaction mixture was
extracted with dichloromethane, washed with water,
and dried over MgSO4. The solvent was evaporated
under reduced pressure to obtain the crude product,
which was purified by column chromatography over
silica gel (PE/EA = 4/1 ∼ 1/1) to give white solid.
General procedure for compounds 5, 6, 11, and 12
Compounds 3, 4, 9, or 10 were dissolved in metha-
nol, 85% hydrazine hydrate was added, then refluxed for
1 h (Fig. 1). The 4 mol/L NaOH of aqueous solution was
added and stirred for 0.5 h, extracted with dichloro-
methane, washed with water and dried. The solvent was
evaporated under reduced pressure to obtain the crude
product which was purified by gel permeation chroma-
tography HW-40C (dichloromethane/methanol = 2/1)
to give a white solid.
(17E )-3β-(1,3-Dioxoisoindolin-2-yl)-pregn-
17(20)-en-16α-ol (9)
(17Z )-Pregn-17(20)-en-3β-amine (5)
1
Yield: 88.3%. 1H NMR (400 MHz, CDCl3): δ 5.10
Yield: 76.4%. H NMR (400 MHz, CDCl3): δ
(qt, J1 = 2.0, J2 = 6.8 Hz, 1H), 2.67 (br s, 1H), 1.64 (dt, 7.82–7.80 (m, 2H), 7.70–7.68 (m, 2H), 5.58 (q,
Drug Dev. Res.

80
WANG ET AL.
J = 7.2 Hz, 1H), 4.43 (d, J = 5.2 Hz, 1H), 4.18 (m, 1H), was stirred in ice bath for 1 h, evaporated just to dryness
1.74 (d, J = 7.2 Hz, 3H), 0.99 (s, 3H), 0.89 (s, 3H). 13C with a vacuum evaporator. NaHCO3 solution was added
NMR (100 MHz, CDCl3): δ 168.5, 155.6, 133.8, 132.1, until the mixture reached neutral (Fig. 1). The reaction
123.0, 119.4, 74.4, 54.1, 52.5, 50.5, 46.1, 44.5, 37.7, mixture was extracted with dichloromethane, washed
37.4, 35.6, 35.1,34.4, 31.8, 31.7, 28.5, 25.1, 21.2, 17.6, with NaHCO3 solution, and dried over MgSO4. The
13.2, 12.2. ESI-MS m/z: 446 [M-H]−.
solvent was evaporated under reduced pressure to
obtain the colorless oil.
(17E )-3α-(1,3-Dioxoisoindolin-2-yl)-pregn-
17(20)-en-16α-ol (10)
(17E )-3β-Aminopregn-17(20)-en-16-one (17)
Yield: 92.6%. 1H NMR (400 MHz, CDCl3): δ 6.48
(q, J = 7.6 Hz, 1H), 2.67 (br s, 1H), 1.84 (d, J = 7.6 Hz,
3H), 1.01 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 206.6, 148.0, 129.0, 54.2, 51.1, 50.1, 45.5,
43.4, 39.3, 38.0, 37.3, 36.4, 35.7, 34.2, 32.5, 32.0, 28.5,
20.9, 17.7, 13.2, 12.3. ESI-MS m/z: 314 [M-H]−.
1
Yield: 71.8%. H NMR (400 MHz, CDCl3): δ
7.82–2.80 (m, 2H), 7.71–7.69 (m, 2H), 5.56 (q,
J = 7.2 Hz, 1H), 4.49 (br s, 1H), 4.41 (d, J = 5.4 Hz,
1H), 1.72 (d, J = 7.2 Hz, 3H), 0.87 (s, 3H), 0.86 (s, 3H).
13C NMR (100 MHz, CDCl3): δ 169.2, 155.6, 133.8,
132.1, 122.9, 119.4, 74.4, 54.5, 52.4, 47.5, 44.5, 40.4,
37.4, 35.1, 34.9, 34.5, 32.7, 31.6, 28.5, 24.6, 20.9, 17.6,
13.2, 12.4. ESI-MS m/z: 446 [M-H]−.
(17E )-3α-Aminopregn-17(20)-en-16-one (18)
Yield: 96.1%. 1H NMR (400 MHz, CDCl3): δ 6.48
(q, J = 7.2 Hz, 1H), 3.24 (br s, 1H), 1.84 (d, J = 7.2 Hz,
3H), 1.01 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 206.6, 148.0, 128.9, 54.1, 50.1, 45.8, 43.4,
39.0, 37.9, 36.5, 36.3, 35.8, 34.2, 31.9, 31.8, 28.7, 28.4,
20.5, 17.7, 13.2, 12.3. ESI-MS m/z: 314 [M-H]−.
General Procedure for Compounds 15 and 16
Compound 13g or 14g (1 eq) was dissolved in
dichloromethane, and activity MnO2 powder (20 eq)
was added. The mixture was refluxed for 2 h, filtered
with Celite (Fig. 1). The solvent was evaporated under
reduced pressure to obtain the crude product, which
was purified by column chromatography over silica gel
(PE/EA = 4/1) to give yellow oil.
General procedure for compounds
7g, 8g, 13g, and 14g
Compound 5, 6, 11, or 12 (1.0 eq) was dissolved
in THF, then TEA (2.0 eq) was added, when the
mixture was stirred in ice bath, and THF solution of
Boc2O (1.2 eq) was added (Fig. 1). The mixture was
then stirred at room temperature for 17 h, evaporated
just to dryness with a vacuum evaporator. The reaction
mixture was extracted with ethyl acetate, washed with
water, then washed with saturated brine and dried over
MgSO4. The solvent was evaporated under reduced
pressure to obtain the crude product, which was puri-
fied by column chromatography over silica gel to give
white solid.
(17E )-3β-(tert-Butoxycarbonylamino)-pregn-
17(20)-en-16-one (15)
Yield: 74.0%. 1H NMR (400 MHz, CDCl3): δ 6.49
(q, J = 7.2 Hz, 1H), 4.40 (br s, 1H), 3.42 (br s, 1H), 1.84
(d, J = 7.2 Hz, 3H), 1.44 (s, 9H), 1.01 (s, 3H), 0.82 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 206.6, 148.0,
129.0, 54.0, 50.0, 45.4, 43.4, 37.9, 37.1, 36.3, 35.7, 35.6,
34.2, 31.9, 29.1, 28.4, 28.3, 20.8, 17.6, 13.2, 12.2.
ESI-MS m/z: 414 [M-H]−.
(17Z )-3β-(tert-Butoxycarbonylamino)-pregn-
17(20)-en (7g)
(17E )-3α-(tert-Butoxycarbonylamino)-pregn-
17(20)-en-16-one (16)
Yield: 87.7%. 1H NMR (400 MHz, CDCl3): δ 5.11
(qt, J1 = 1.9, J2 = 7.1 Hz, 1H), 4.37 (s, 1H), 3.41 (br s,
1H), 1.64 (dt, J1 = 1.9, J2 = 7.1 Hz, 3H), 1.44 (s, 9H),
0.86 (s, 3H), 0.78 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 155.3, 150.4, 113.2, 79.0, 56.2, 54.3, 50.1, 45.4, 44.3,
37.4, 37.2, 35.8, 35.5, 35.0, 31.8, 31.4, 29.2, 28.5, 28.4,
24.4, 21.3, 16.9, 13.1, 12.2. ESI-MS m/z: 400 [M-H]−.
Yield: 75.6%. 1H NMR (400 MHz, CDCl3): δ 6.49
(q, J = 7.2 Hz, 1H), 4.83 (br s, 1H), 3.85 (br s, 1H), 1.84
(d, J = 7.2 Hz, 3H), 1.45 (s, 9H), 1.01 (s, 3H), 0.84 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 206.5, 155.2,
147.9, 129.0, 79.0, 54.2, 50.1, 45.7, 43.4, 40.6, 37.9,
36.3, 36.2, 34.1, 33.2, 32.7, 31.9, 28.5, 28.2, 26.3, 20.5,
17.6, 13.2, 11.4. ESI-MS m/z: 414 [M-H]−.
(17Z )-3α-(tert-Butoxycarbonylamino)-pregn-
17(20)-en (8g)
General procedure for compounds 17 and 18
Compound 15 or 16 was dissolved in dichloro-
Yield: 82.5%. 1H NMR (400 MHz, CDCl3): δ 5.11
methane, CF3COOH was added, and then the mixture (qt, J1 = 2.0 Hz, J2 = 7.1 Hz, 1H), 3.84 (s, 1H), 1.65
Drug Dev. Res.

ANTIMETASTATIC E-SALIGNONE AMIDE DERIVATIVES
81
(dt, J1 = 2.0 Hz, J2 = 7.1 Hz, 3H), 1.45 (s, 9H), 0.86 (17Z )-3α-Acetamidopregn-17(20)-en (8a)
(s, 3H), 0.80 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
Yield: 90.0%. 1H NMR (400 MHz, CDCl3): δ 5.84
150.4, 113.2, 56.4, 54.5, 44.4, 37.2, 36.1, 35.0, 33.3, (br s, 1H), 5.10 (qt, J1 = 1.9, J2 = 7.2 Hz, 1H), 4.10 (d,
31.8, 31.4, 28.5, 26.4, 24.3, 21.0, 16.9, 13.1, 11.4. J = 7.0 Hz, 1H), 1.98 (s, 3H), 1.64 (dt, J1 = 1.9,
ESI-MS m/z: 400 [M-H]−.
J2 = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 169.2,
150.4, 113.3, 56.4, 54.6, 44.8, 44.4, 41.0, 37.2, 36.1,
35.0, 33.2, 32.8, 31.8, 31.4, 28.5, 28.4, 26.0, 24.3, 23.7,
21.0, 16.9, 13.1, 11.4. ESI-MS m/z: 342 [M-H]−.
(17E )-3β-(tert-Butoxycarbonylamino)-pregn-
17(20)-en-16α-ol (13g)
Yield: 80.1%. 1H NMR (400 MHz, CDCl3): δ 5.57
(q, J = 6.8 Hz, 1H), 4.41 (d, J = 4.8 Hz, 1H), 3.41 (br s,
1H), 1.73 (d, J = 6.8 Hz, 3H), 1.73 (d, J = 6.8 Hz, 3H),
1.44 (s, 9H), 0.85 (s, 3H), 0.78 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 155.5, 119.4, 74.4, 54.3, 52.5,
45.4, 44.5, 37.4, 35.8, 35.5, 35.1, 34.4, 31.7, 29.2, 28.5,
21.2, 17.6, 13.2, 12.2. ESI-MS m/z: 416 [M-H]−.
(17Z )-3β-Pivalamidopregn-17(20)-en (7c)
Yield: 85.7%. 1H NMR (400 MHz, CDCl3): δ 5.41
(d, J = 7.6 Hz, 1H), 5.11 (q, J = 7.1 Hz, 1H), 3.74 (m,
1H), 1.65 (d, J = 7.1 Hz, 3H), 1.18 (s, 9H), 0.87 (s, 3H),
0.82 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 177.5,
150.4, 113.2, 56.2, 54.3, 48.7, 45.3, 44.3, 38.5, 37.4,
37.2, 35.5, 35.4, 35.1, 31.8, 31.4, 28.8, 28.5, 27.6, 24.4,
21.3, 16.9, 13.1, 12.2. ESI-MS m/z: 384 [M-H]−.
(17E )-3α-(tert-Butoxycarbonylamino)-pregn-
17(20)-en-16α-ol (14g)
Yield: 86.6%. 1H NMR (400 MHz, CDCl3): δ 5.58
(q, J = 7.1 Hz, 1H), 4.42 (d, J = 4.9 Hz, 1H), 3.84 (br s,
1H), 1.45 (s, 9H), 0.85 (s, 3H), 0.81 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 155.5, 155.3, 119.4, 74.3, 54.5,
52.5, 44.5, 40.7, 37.4, 36.1, 35.1, 34.4, 33.3, 33.0, 31.8,
28.5, 28.4, 26.4, 20.8, 17.6, 13.2, 11.4. ESI-MS m/z: 416
[M-H]−.
(17Z )-3α-Pivalamidopregn-17(20)-en (8c)
Yield: 82.3%. 1H NMR (400 MHz, CDCl3): δ 5.91
(d, J = 5.8 Hz, 1H), 5.12 (qt, J1 = 1.8, J2 = 7.2 Hz, 1H),
4.09 (br s, 1H), 1.65(dt, J1 = 1.8, J2 = 7.2 Hz, 3H), 1.21
(s, 9H), 0.87 (s, 3H), 0.82 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 177.4, 150.3, 113.3, 56.3, 54.7, 44.3, 41.4,
38.7, 37.2, 36.1, 35.0, 33.4, 32.8, 31.8, 31.4, 28.5, 27.7,
25.9, 24.3, 21.0, 16.9, 13.1, 11.4. ESI-MS m/z: 384
[M-H]−.
General Procedure for Compounds 7a, 8a, 7c, 8c,
13c, 14c, 19a, 20a, 19c, and 20c
(17E )-3β-Acetamidopregn-17(20)-en-16-one (19a)
Yield: 92.2%. 1H NMR (400 MHz, CDCl3): δ 6.49
(q, J = 7.5 Hz, 1H), 5.30 (d, J = 8.1 Hz, 1H), 3.76 (m,
1H), 1.96 (s, 3H), 1.85 (d, J = 7.5 Hz, 3H), 1.01 (s, 3H),
0.84 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 206.5,
169.2, 148.0, 129.0, 54.0, 49.9, 48.9, 45.3, 43.4, 37.9,
37.0, 36.3, 35.6, 35.3, 34.2, 31.8, 28.8, 28.3, 23.6, 20.8,
17.7, 13.1, 12.2. ESI-MS m/z: 356 [M-H]−.
The compound 5, 6, 11, 12, 17, or 18 (1 eq) was
dissolved in CH2Cl2, then DMAP (1.2 eq) was added
at room temperature. The acid chloride (1.2 eq) was
added into the mixture. The mixture was stirred at room
temperature for 24 h, and then evaporated just to
dryness with a vacuum evaporator. The reaction
mixture was extracted with CH2Cl2, washed with water,
then washed with saturated brine and dried over
MgSO4. The solvent was evaporated under reduced
pressure to obtain the crude product which was purified
by column chromatography over silica gel to give white
solid.
(17E )-3α-Acetamidopregn-17(20)-en-16-one (20a)
Yield: 86.5%. 1H NMR (400 MHz, CDCl3) δ 6.50
(q, J = 7.6 Hz, 1H), 5.75 (br s, 1H), 4.14 (br s, 1H), 2.00
(s, 3H), 1.85 (d, J = 7.6 Hz, 3H), 1.02 (s, 3H), 0.86 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 206.3, 169.2,
(17Z )-3β-Acetamidopregn-17(20)-en (7a)
Yield: 87.4%. 1H NMR (400 MHz, CDCl3): δ 5.67 147.9, 129.0, 65.5, 54.3, 50.2, 44.7, 43.4, 41.0, 37.9,
(d, J = 8.0 Hz, 1H), 5.10 (qt, J1 = 2.0, J2 = 7.2 Hz, 1H), 36.4, 36.2, 34.1, 32.9, 32.7, 31.9, 28.2, 25.9, 23.7, 20.5,
3.74 (m, 1H), 1.95 (s, 3H), 1.64 (dt, J1 = 2.0, J2 = 7.2 Hz, 17.6, 13.1, 11.4. ESI-MS m/z: 357 [M-H]−
3H), 0.86 (s, 3H), 0.80 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 169.3, 150.3, 113.2, 56.2, 54.3, 49.0, 45.3,
44.3, 37.3, 37.2, 35.5, 35.4, 35.0, 31.8, 31.4, 28.8, 28.5, (17E )-3β-Pivalamidopregn-17(20)-en-16-one (19c)
24.4, 23.5, 21.3, 16.9, 13.1, 12.2. ESI-MS m/z: 342
Yield: 86.7%. 1H NMR (400 MHz, CDCl3): δ 6.49
[M-H]−.
(q, J = 7.5 Hz, 1H), 5.42 (d, J = 7.9 Hz, 1H), 3.74
Drug Dev. Res.

82
WANG ET AL.
(m, 1H), 1.84 (d, J = 7.5 Hz, 3H), 1.18 (s, 9H), 1.01 (s, 165.0, 150.2, 139.1, 132.4, 127.7, 118.1, 114.8, 113.3,
3H), 0.85 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 206.6, 56.3, 54.6, 45.7, 44.3, 41.3, 37.2, 36.2, 34.9, 33.4, 32.7,
177.6, 148.0, 129.0, 53.9, 50.0, 48.6, 45.3, 43.4, 38.5, 31.8, 31.4, 28.4, 25.9, 24.3, 21.0, 16.9, 13.1, 11.4.
37.9, 37.1, 36.3, 35.6, 35.3, 34.2, 31.9, 28.7, 28.3, 27.6, ESI-MS m/z: 429 [M-H]−.
20.8, 17.7, 13.2, 12.3. ESI-MS m/z: 398 [M-H]−.
(17Z )-3β-Cinnamamidopregn-17(20)-en (7d)
Yield: 67.5%. 1H NMR (400 MHz, CDCl3): δ 7.60
(17E )-3α-Pivalamidopregn-17(20)-en-16-one (20c)
Yield: 88.6%. 1H NMR (400 MHz, CDCl3): δ 6.50
(d, J = 15.6 Hz, 1H), 7.49–7.47 (m, 2H), 7.36–7.33 (m,
3H), 6.36 (d, J = 15.6 Hz, 1H), 5.54 (d, J = 8.0 Hz, 1H),
5.11 (d, J1 = 1.8, J2 = 7.6 Hz, 1H), 3.91 (m, 1H), 1.65 (d,
J1 = 1.8, J2 = 7.6 Hz, 1H), 0.87 (s, 3H), 0.82 (s, 3H). 13C
NMR (100 MHz, CDCl3): δ 165.0, 150.4, 140.7, 135.0,
129.5, 128.8, 127.7, 121.2, 113.2, 56.2, 54.3, 49.2, 45.3,
44.4, 37.4, 37.2, 35.5, 35.1, 31.8, 31.4, 29.7, 28.9, 28.6,
24.4, 21.3, 16.9, 13.1, 12.2. ESI-MS m/z: 430 [M-H]−.
(q, J = 7.6 Hz, 1H), 5.89 (d, J = 5.2 Hz, 1H), 4.10 (br s,
1H), 1.85 (d, J = 7.6 Hz, 3H), 1.21 (s, 9H), 1.02 (s, 3H),
0.86 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 206.4,
177.4, 147.9, 129.0, 54.4, 50.2, 44.2, 43.4, 41.4, 38.8,
37.9, 36.3, 36.2, 34.2, 33.2, 32.7, 31.9, 28.3, 27.7, 25.8,
20.6, 17.7, 13.2, 11.4. ESI-MS m/z: 398 [M-H]−.
General procedures for compounds 7b, 7d–f, 8b,
8d–f, 13a–b, 13d–f, 14a–b, 14d–f, 19b, 19d–f, 20b,
and 20d–f
(17Z )-3α-Cinnamamidopregn-17(20)-en (8d)
Yield: 53.9%. 1H NMR (400 MHz, CDCl3): δ 7.63
(d, J = 15.6 Hz, 1H), 7.52–7.50 (m, 2H), 7.37–7.35 (m,
3H), 6.44 (d, J = 15.6 Hz, 1H), 5.90 (d, J = 7.0 Hz, 1H),
5.12 (d, J = 7.1 Hz, 1H), 4.29 (br s, 1H), 1.65 (d,
J = 7.1 Hz, 1H), 0.87 (s, 3H), 0.84 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 165.0, 150.4, 140.8, 135.0, 130.0,
128.8, 127.8, 121.2, 113.3, 56.4, 54.6, 45.0, 44.4, 41.1,
37.2, 36.2, 35.0, 33.3, 32.8, 31.8, 31.4, 28.4, 26.0, 24.3,
21.0, 16.9, 13.1, 11.5. ESI-MS m/z: 430 [M-H]−.
Acid (1.0 eq) was dissolved in CH2Cl2, then CDI
(1.2 eq) was added, when the mixture was stirred in ice
bath for 2 h. The amine (1.2 eq), such as compound 5,
6, 11, 12, 17 or 18, was added into the mixture. The
mixture was stirred at room temperature for 24 h, and
then evaporated just to dryness with a vacuum evapo-
rator. The reaction mixture was extracted with CH2Cl2,
washed with water, then washed with saturated brine
and dried over MgSO4. The solvent was evaporated
under reduced pressure to obtain the crude product
which was purified by column chromatography over
silica gel to give white solid.
(17Z )-3β-(3, 4-Diclorobenzamido)-pregn-17(20)-en
(7e)
Yield: 88.2%. 1H NMR (400 MHz, CDCl3): δ 7.84
(s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 8.3 Hz,
1H), 6.20 (d, J = 6.9 Hz, 1H), 5.11 (q, J = 7.1 Hz, 1H),
3.92 (br s, 1H), 1.65 (d, J = 7.1 Hz, 3H), 0.86 (s, 3H),
0.80 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 163.5,
149.3, 134.6, 133.8, 131.9, 129.5, 128.1, 125.1, 112.3,
55.2, 53.2, 48.8, 44.3, 43.3, 36.3, 36.2, 34.5, 34.0, 30.7,
30.4, 27.7, 27.5, 23.4, 21.7, 20.3, 15.9, 13.1, 11.2.
ESI-MS m/z: 473 [M-H]−.
(17Z )-3β-Paracyanobenzamidopregn-17(20)-en
(7b)
Yield: 82.2%. 1H NMR (400 MHz, CDCl3): δ 7.87
(d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.1 Hz, 2H), 6.07 (d,
J = 7.8 Hz, 1H), 5.14 (q, J = 7.2 Hz, 1H), 3.98 (br s,
1H), 1.67 (d, J = 7.1 Hz, 3H), 0.89 (s, 3H), 0.85 (s, 3H).
13C NMR (100 MHz, CDCl3): δ 164.9, 150.3, 139.0,
132.4, 127.6, 118.0, 114.9, 113.3, 56.2, 54.3, 49.9, 45.3,
44.3, 37.3, 37.2, 35.5, 35.3, 35.1, 31.8, 31.4, 29.7, 28.5,
24.4, 21.3, 16.9, 13.1, 12.2. ESI-MS m/z: 429 [M-H]−.
(17Z )-3α-(3,4-Diclorobenzamido)-pregn-17(20)-en
(8e)
Yield: 85.2%. 1H NMR (400 MHz, CDCl3): δ 7.85
(d, J = 1.9 Hz, 1H), 7.59 (dd, J1 = 1.9 Hz, J1 = 8.3 Hz
1H), 7.52 (d, J = 8.3 Hz, 1H), 6.28 (d, J = 6.0 Hz, 1H),
5.12 (q, J = 7.2 Hz, 1H), 4.31 (br s, 1H), 1.65 (d,
(17Z )-3α-Paracyanobenzamidopregn-17(20)-en
(8b)
Yield: 76.3%. 1H NMR (400 MHz, CDCl3): δ 7.86 J = 7.2 Hz, 3H), 0.88 (s, 3H), 0.86 (s, 3H). 13C NMR
(d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.4 Hz, 2H), 6.44 (d, (100 MHz, CDCl3): δ 164.5, 150.3, 135.7, 135.0, 133.1,
J = 7.1 Hz, 1H), 5.11 (qt, J1 = 1.8, J2 = 7.1 Hz, 1H), 4.32 130.6, 129.1, 126.1, 113.3, 56.3, 54.6, 45.6, 44.4, 41.3,
(br s, 1H), 1.64 (dt, J1 = 1.8, J2 = 7.1 Hz, 3H), 0.87 (s, 37.2, 36.2, 35.0, 33.4, 32.7, 31.7, 31.4, 28.4, 26.0, 24.3,
3H), 0.85 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 21.0, 16.9, 13.1, 11.4. ESI-MS m/z: 473 [M-H]−.
Drug Dev. Res.

ANTIMETASTATIC E-SALIGNONE AMIDE DERIVATIVES
83
(17Z )-3β-Benzamidopregn-17(20)-en (7f )
71.2, 54.7, 51.6, 49.8, 45.5, 44.1, 37.3, 36.7, 35.9, 35.7,
1
Yield: 82.7%. H NMR (400 MHz, CDCl3): δ 35.3, 34.7, 31.8, 28.8, 28.5, 21.1, 20.6, 14.6, 12.3.
7.76–7.74 (m, 2H), 7.48–7.40 (m, 3H), 5.96 (d, ESI-MS m/z: 445 [M-H]−.
J = 7.8 Hz, 1H), 5.12 (qt, J1 = 1.9, J2 = 7.1 Hz, 1H), 3.98
(m, 1H), 1.65 (dt, J1 = 1.9, J2 = 7.1 Hz, 3H), 0.87 (s,
(17E )-3α-Paracyanobenzamidopregn-17(20)-en-
3H), 0.84 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
16α-ol (14b)
Yield: 64.7%. 1H NMR (400 MHz, CDCl3): δ 7.85
166.8, 150.4, 135.1, 133.5, 131.3, 130.1, 128.5, 128.4,
126.8, 113.2, 56.2, 54.3, 49.4, 45.4, 44.4, 37.4, 37.2,
35.6, 35.5, 35.1, 31.8, 31.4, 28.9, 28.6, 24.4, 21.3, 16.9,
13.1, 12.3. ESI-MS m/z: 420 [M-H]−.
(d, J = 8.3 Hz, 2H), 7.72 (d, J = 8.3 Hz, 2H), 6.42 (d,
J = 7.1 Hz, 1H), 5.56 (q, J = 7.1 Hz, 1H), 4.40 (d,
J = 5.0 Hz, 1H), 4.31 (br s, 1H),1.71 (d, J = 7.1 Hz, 3H),
0.84 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 165.0,
155.3, 139.1, 132.4, 127.7, 119.6, 118.1, 114.9, 113.3,
74.3, 54.6, 52.5, 45.7, 44.5, 41.3, 37.3, 36.2, 35.0, 34.3,
33.3, 32.7, 31.7, 28.3, 25.9, 20.9, 17.6, 13.2, 11.4.
ESI-MS m/z: 445 [M-H]−.
(17Z )-3α-Benzamidopregn-17(20)-en (8f)
1
Yield: 79.4%. H NMR (400 MHz, CDCl3) δ
7.79–7.76 (m, 2H), 7.52–7.42 (m, 3H), 6.41 (d,
J = 6.6 Hz, 1H), 5.12 (qt, J1 = 1.6, J2 = 7.2 Hz, 1H), 4.34
(m, 1H), 1.65 (dt, J1 = 1.6, J2 = 7.2 Hz, 3H), 0.88 (s,
3H), 0.86 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
166.8, 150.4, 135.2, 131.3, 130.1, 128.6, 128.4, 126.9,
113.3, 56.3, 54.6, 45.3, 44.4, 41.3, 37.2, 36.2, 35.0, 33.4,
32.8, 31.8, 31.4, 28.5, 26.1, 24.3, 21.0, 16.9, 13.1, 11.5.
ESI-MS m/z: 420 [M-H]−.
(17E )-3β-Pivalamidopregn-17(20)-en-16α-ol (13c)
Yield: 50.6%. 1H NMR (400 MHz, CDCl3) δ 5.41
(d, J = 8.0 Hz, 1H), 5.30 (q, J = 6.8 Hz, 1H), 4.77 (d,
J = 5.6 Hz, 1H), 3.73 (m, 1H), 1.78 (d, J = 6.8 Hz, 3H),
1.17 (s, 9H), 0.82 (s, 3H), 0.72 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 177.6, 156.2, 117.5, 71.2, 54.6,
51.6, 48.7, 45.5, 44.1, 38.5, 37.4, 36.7, 36.0, 35.7, 35.4,
34.7, 31.9, 28.8, 28.5, 27.6, 21.1, 20.6, 14.6, 12.3.
ESI-MS m/z: 400 [M-H]−.
(17E )-3β-Acetamidopregn-17(20)-en-16α-ol (13a)
Yield: 62.5%. 1H NMR (400 MHz, CDCl3): δ 5.58
(dd, J1 = 1.1, J2 = 7.2 Hz, 1H), 5.35 (d, J = 7.5 Hz, 1H),
4.43 (d, J = 5.2 Hz, 1H), 3.77 (m, 1H), 1.96 (s, 3H), 1.74
(dd, J1 = 1.1, J2 = 7.2 Hz, 3H), 0.87 (s, 3H), 0.82 (s, 3H).
13C NMR (100 MHz, CDCl3): δ 169.2, 155.5, 119.4,
74.4, 54.3, 52.4, 48.9, 45.3, 44.5, 37.4, 37.3, 35.5, 35.4,
35.0, 34.4, 31.7, 28.9, 28.4, 23.6, 21.2, 17.6, 13.2, 12.2.
ESI-MS m/z: 358 [M-H]−.
(17E )-3α-Pivalamidopregn-17(20)-en-16α-ol (14c)
Yield: 57.8%. 1H NMR (400 MHz, CDCl3): δ 5.91
(d, J = 6.1 Hz, 1H), 5.59 (q, J = 7.2 Hz, 1H), 4.43 (d,
J = 5.0 Hz, 1H), 4.10 (br s, 1H), 1.74 (d, J = 7.2 Hz,
3H), 1.22 (s, 9H), 0.87 (s, 3H), 0.83 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 177.4, 155.4, 119.5, 74.4, 54.7,
52.6, 44.5, 44.3, 41.4, 37.4, 36.2, 35.0, 34.3, 33.4, 32.7,
31.7, 28.4, 27.7, 25.9, 20.9, 17.6, 13.2, 11.4. ESI-MS
m/z: 400 [M-H]−.
(17E )-3α-Acetamidopregn-17(20)-en-16α-ol (14a)
Yield: 57.2%. 1H NMR (400 MHz, CDCl3): δ 5.73
(br s, 1H), 5.35 (qd, J1 = 1.6, J2 = 7.2 Hz, 1H), 4.79 (d,
J = 5.2 Hz, 1H), 4.13 (s, 1H), 1.99 (s, 3H), 1.78 (d,
J = 7.2 Hz, 3H), 0.82 (s, 3H), 0.73 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 169.2, 156.2, 117.5, 71.1, 55.0,
51.7, 44.7, 44.1, 41.1, 36.7, 36.2, 35.9, 34.6, 33.2, 32.8,
31.9, 28.4, 26.0, 23.7, 20.8, 20.6, 14.6, 11.4. ESI-MS
m/z: 358 [M-H]−.
(17E )-3β-Cinnamamidopregn-17(20)-en-16α-ol
(13d)
Yield: 47.0%. 1H NMR (400 MHz, CDCl3): δ 7.77
(d, J = 16.0 Hz, 1H), 7.50 (m, 5H), 6.46 (d, J = 16.0 Hz,
1H), 6.34 (d, J = 15.6 Hz, 1H), 5.58 (q, J = 7.2 Hz, 1H),
5.42 (d, J = 8.0 Hz, 1H), 4.42 (d, J = 5.2 Hz, 1H), 3.91
(m, 1H), 1.73 (d, J = 7.2 Hz, 1H), 0.86 (s, 3H), 0.83 (s,
3H). 13C NMR (100 MHz, DMSO): δ 165.0, 155.4,
140.8, 135.0, 129.5, 128.8, 127.8, 121.2, 119.4, 74.4,
54.6, 52.6, 44.9, 44.5, 41.4, 37.4, 36.2, 35.0, 34.4, 33.2,
32.9, 31.8, 28.4, 26.0, 20.9, 17.6, 13.2, 11.4. ESI-MS
m/z: 446 [M-H]−.
(17E )-3β-Paracyanobenzamidopregn-17(20)-en-
16α-ol (13b)
Yield: 61.8%. 1H NMR (400 MHz, CDCl3) δ 7.86
(d, J = 8.0 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H), 6.09 (d,
J = 7.9 Hz, 1H), 5.33 (q, J = 7.0 Hz, 1H), 4.79 (d, (17E )-3α-Cinnamamidopregn-17(20)-en-16α-ol
J = 5.5 Hz, 1H), 3.98 (m, 1H), 1.79 (d, J = 7.0 Hz, 3H), (14d)
1
0.85 (s, 3H), 0.75 (s, 3H). 13C NMR (100 MHz, CDCl3):
Yield: 46.8%. H NMR (400 MHz, DMSO): δ
δ 164.9, 156.2, 139.0, 132.4, 127.6, 118.0, 117.6, 114.9, 7.63 (d, J = 15.6 Hz, 1H), 7.53–7.51 (m, 2H), 7.39–7.34
Drug Dev. Res.

84
WANG ET AL.
(m, 3H), 6.44 (d, J = 15.6 Hz, 1H), 5.92 (d, J = 7.3 Hz, J = 7.1 Hz, 3H), 0.87 (s, 3H), 0.86 (s, 3H). 13C NMR
1H), 5.58 (q, J = 6.8 Hz, 1H), 4.43 (d, J = 5.2 Hz, 1H), (100 MHz, CDCl3): δ 166.7, 155.4, 135.3, 131.3, 128.6,
4.31 (m, 1H), 1.74 (d, J = 6.8 Hz, 3H), 0.87 (s, 3H), 0.84 126.9, 119.5, 54.6, 52.5, 45.2, 44.5, 41.3, 37.4, 36.2,
(s, 3H). 13C NMR (100 MHz, DMSO): δ 165.0, 155.4, 35.0, 34.3, 33.4, 32.8, 31.7, 28.4, 26.0, 20.9, 17.6, 13.2,
140.8, 135.0, 129.5, 128.8, 127.8, 121.2, 119.4, 74.4, 11.4. ESI-MS m/z: 420 [M-H]−.
54.6, 52.6, 44.9, 44.5, 41.4, 37.4, 36.2, 35.0, 34.4, 33.2,
32.9, 31.8, 28.4, 26.0, 20.9, 17.6, 13.2, 11.4. ESI-MS
m/z: 446 [M-H]−.
(17E )-3β-Paracyanobenzamidopregn-17(20)-en-16-
one (19b)
Yield: 79.5%. 1H NMR (400 MHz, CDCl3): δ 7.85
(17E )-3β-(3,4-Diclorobenzamido)-pregn-17(20)-en-
16α-ol (13e)
(d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 6.49 (q,
J = 7.5 Hz, 1H), 6.00 (d, J = 7.9 Hz, 1H), 4.00 (m, 1H),
1.85 (d, J = 7.5 Hz, 3H), 1.02 (s, 3H), 0.88 (s, 3H). 13C
NMR (100 MHz, CDCl3): δ 206.4, 164.9, 147.9, 138.9,
132.4, 129.0, 127.6, 53.9, 50.0, 49.8 45.3, 43.4, 37.9,
37.0, 36.3, 35.7, 35.2, 34.2, 31.8, 28.7, 28.3, 20.9, 17.7,
13.2, 12.3. ESI-MS m/z: 443 [M-H]−.
Yield: 44.7%. 1H NMR (400 MHz, CDCl3): δ 7.83
(s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 8.4 Hz,
1H), 5.96 (d, J = 7.6 Hz, 1H), 5.33 (q, J = 6.8 Hz, 1H),
4.78 (d, J = 5.6 Hz, 1H), 3.93 (br s, 1H), 1.78 (d,
J = 6.8 Hz, 3H), 0.83 (s, 3H), 0.73 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 164.5, 156.2, 135.7, 134.8, 133.0,
130.6, 129.1, 126.1, 117.6, 71.2, 54.6, 51.6, 49.7, 45.5,
44.2, 37.3, 36.7, 35.9, 35.7, 35.3, 34.7, 31.9, 28.8, 28.5,
21.1, 20.6, 14.6, 12.3. ESI-MS m/z: 489 [M-H]−.
(17E )-3α-Paracyanobenzamidopregn-17(20)-en-16-
one (20b)
Yield: 74.2%. 1H NMR (400 MHz, CDCl3): δ 7.88
(d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.0 Hz, 2H), 6.50 (q,
J = 7.4 Hz, 1H), 4.36 (m, 1H), 1.84 (d, J = 7.4 Hz, 3H),
1.03 (s, 3H), 0.90 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 206.3, 165.0, 147.8, 139.1, 132.4, 129.2, 127.7, 118.1,
114.9, 54.3, 50.2, 45.7, 43.3, 41.2, 37.9, 36.2, 34.1, 33.0,
32.7, 31.9, 29.7, 28.2, 25.9, 20.6, 17.7, 13.2, 11.5.
ESI-MS m/z: 443 [M-H]−.
(17E )-3α-(3,4-Diclorobenzamido)-pregn-17(20)-en-
16α-ol (14e)
Yield: 45.1%. 1H NMR (400 MHz, CDCl3): δ 7.85
(d, J = 1.9 Hz, 1H), 7.59 (dd, J1 = 1.9 Hz, J1 = 8.2 Hz,
1H), 7.52 (d, J = 8.3 Hz, 1H), 6.29 (d, J = 6.7 Hz, 1H),
5.32 (q, J = 6.9 Hz, 1H), 4.79 (d, J = 5.6 Hz, 1H), 4.31
(br s, 1H), 1.78 (d, J = 6.9 Hz, 3H), 0.86 (s, 3H), 0.74 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 164.6, 156.1,
135.7, 135.0, 133.1, 130.6, 129.1, 126.1, 117.6, 71.2,
54.9, 51.7, 45.6, 44.2, 41.4, 36.7, 36.3, 35.8, 34.6, 33.4,
32.7, 31.8, 28.4, 25.9, 20.8, 20.6, 14.6, 11.5. ESI-MS
m/z: 489 [M-H]−.
(17E )-3β-Cinnamamidopregn-17(20)-en-16-one
(19d)
Yield: 72.9%. 1H NMR (400 MHz, CDCl3): δ 7.61
(d, J = 15.6 Hz, 1H), 7.51–7.49 (m, 2H), 7.36–7.35 (m,
3H), 6.49 (q, J = 7.5 Hz, 1H), 6.35 (d, J = 15.6 Hz, 1H),
5.48 (d, J = 7.6 Hz, 1H), 3.94 (m, 1H), 1.85 (d,
J = 7.5 Hz, 3H), 1.01 (s, 3H), 0.86 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 206.6, 165.1, 148.0, 140.9, 134.9,
130.9, 129.6, 129.0, 128.8, 127.8, 120.9, 54.0, 50.0, 49.1,
45.2, 43.4, 37.9, 37.0, 36.3, 35.6, 35.3, 34.2, 31.8, 28.8,
28.3, 20.8, 17.7, 13.2, 12.2. ESI-MS m/z: 444 [M-H]−.
(17E )-3β-Benzamidopregn-17(20)-en-16α-ol (13f)
1
Yield: 52.9%. H NMR (400 MHz, CDCl3): δ
7.75–7.73 (m, 2H), 7.50–7.39 (m, 3H), 6.02 (d,
J = 8.0 Hz, 1H), 5.59 (qd, J1 = 1.1, J2 = 7.2 Hz, 1H),
4.42 (d, J = 5.3 Hz, 1H), 3.97 (m, 1H), 1.72 (dd,
J1 = 1.1, J2 = 7.2 Hz, 3H), 0.85 (s, 3H), 0.82 (s, 3H). 13C
NMR (100 MHz, CDCl3): δ 166.8, 155.4, 135.0, 131.3,
128.5, 126.8, 119.4, 74.4, 54.3, 52.4, 49.4, 45.4, 44.5,
37.4, 37.3, 35.6, 35.4, 35.1, 34.4, 31.7, 28.9 28.5, 21.2, (17E )-3α-Cinnamamidopregn-17(20)-en-16-one
17.6, 13.2, 12.2. ESI-MS m/z: 420 [M-H]−.
(20d)
Yield: 42.2%. 1H NMR (400 MHz, CDCl3) δ 7.63
(d, J = 15.6 Hz, 1H), 7.51–7.49 (m, 2H), 7.37–7.34 (m,
3H), 6.50 (q, J = 7.5 Hz, 1H), 6.48 (d, J = 15.6 Hz, 1H),
(17E )-3α-Benzamidopregn-17(20)-en-16α-ol (14f)
1
Yield: 50.3%. H NMR (400 MHz, CDCl3): δ 6.08 (br s, 1H), 4.31 (t, J = 6.7 Hz, 1H), 1.85 (d,
7.78–7.76 (m, 2H), 7.52–7.43 (m, 3H), 6.38 (d, J = J = 7.5 Hz, 3H), 1.02 (s, 3H), 0.87 (s, 3H). 13C NMR
6.8 Hz, 1H), 5.58 (q, J = 7.1 Hz, 1H), 4.42 (d, (100 MHz, CDCl3): δ 206.5, 165.1, 148.0, 140.7, 135.0,
J = 5.2 Hz, 1H), 4.34 (t, J = 3.2 Hz, 1H), 1.74 (d, 130.9, 129.5, 129.2, 128.8, 127.7, 121.2, 54.3, 50.2, 44.9,
Drug Dev. Res.

ANTIMETASTATIC E-SALIGNONE AMIDE DERIVATIVES
85
43.4, 41.0, 37.9, 36.3, 36.2, 34.1, 33.0, 32.8, 31.9, 28.2, Invasion assay
26.0, 20.5, 17.7, 13.2, 11.5. ESI-MS m/z: 444 [M-H]−.
The chemotaxis invasion assay was performed as
reported as previously [Zhai et al., 2012]. Briefly, after
pretreatment with the different concentrations of com-
pounds for 24 h, MDA-MB-231 cells suspended in
binding medium (RPMI 1640, 0.1% bovine serum
albumin) were loaded into the upper chambers.
Chemoattractant (EGF, 1 ng/mL) was loaded into the
lower chemotaxis chamber. The 8 μm filter membranes,
which had previously been pretreated with 0.001%
fibronectin, were inserted between the upper and lower
chambers. After further incubation for 3.5 h, the filter
membrane was rinsed, fixed, and stained. The number
of migrating cells was counted under a microscope
(Olympus Corp., CKX41, Tokyo, Japan). The inhibitory
ratio (IR) was calculated as follows: IR% = (1 − number
of migrated cells in sample/number of migrated cells in
control) × 100%, and the median inhibitory concentra-
tion (IC50) was obtained. Data were analyzed by using
SPSS 16.0 software package. LY294002 (Camarillo, CA,
USA), and a phosphoinositide 3-kinase (PI3K) inhibitor
[Kong and Yamori, 2009] reported to inhibit metastasis
[Sun et al., 2005] was used as a positive control.
(17E )-3β-(3,4-Diclorobenzamido)-pregn-17(20)-en-
16-one (19e)
Yield: 43.8%. 1H NMR (400 MHz, CDCl3): δ 7.83
(d, J = 2.0 Hz, 1H), 7.56 (dd, J1 = 2.0, J2 = 8.4 Hz, 1H),
7.50 (d, J = 8.4 Hz, 1H), 6.50 (q, J = 7.6 Hz, 1H), 5.85
(d, J = 8.0 Hz, 1H), 3.96 (m, 1H), 1.85 (d, J = 7.6 Hz,
3H), 1.02 (s, 3H), 0.88 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 206.4, 164.5, 147.9, 135.7, 134.8, 133.0,
130.6, 129.0, 126.1, 54.0, 50.0, 49.7, 45.3, 43.4, 37.9,
37.0, 36.3, 35.7, 35.3, 34.2, 31.8, 28.8, 28.3, 20.9, 17.7,
13.2, 12.3. ESI-MS m/z: 487 [M-H]−.
(17E )-3á-(3,4-Diclorobenzamido)-pregn-17(20)-en-
16-one (20e)
Yield: 48.1%.1H NMR (400 MHz, CDCl3): δ 7.91
(d, J = 2.0 Hz, 1H), 7.64 (dd, J1 = 2.0, J2 = 8.4 Hz, 1H),
7.49 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 7.2 Hz, 1H), 6.49
(q, J = 7.6 Hz, 1H), 4.35 (br s, 1H), 1.85 (d, J = 7.6 Hz,
3H), 1.03 (s, 3H), 0.88 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 206.4, 164.6, 147.9, 135.6, 134.9, 132.8,
130.5, 129.3, 129.1, 126.4, 54.2, 50.3, 45.5, 43.4, 41.0,
37.9, 36.3, 36.2, 34.1, 32.9, 32.7, 31.9, 29.7, 28.1, 25.9,
20.5, 17.7, 13.2, 11.4. ESI-MS m/z: 487 [M-H]−.
Wound healing assay
The assay was carried out as previously reported
[Kong and Yamori, 2010]. MDA-MB-231 cells were
seeded in 6-well plates and grown to full confluence.
The cell monolayer was wounded with a sterile micro-
pipette tip to generate a clean wound area across
the center of the well. Subsequently, cellular debris
was rinsed with PBS, and then incubated in medium
with or without compound for 24 h. Cell migration into
the wound was monitored and photographed under
microscope.
(17E )-3β-Benzamidopregn-17(20)-en-16-one (19f)
1
Yield: 55.8%. H NMR (400 MHz, CDCl3): δ
7.75–7.74 (m, 2H), 7.49–7.41 (m, 3H), 6.49 (q,
J = 7.6 Hz, 1H), 5.95 (d, J = 7.6 Hz, 1H), 3.99 (m, 1H),
1.85 (d, J = 7.6 Hz, 3H), 1.02 (s, 3H), 0.88 (s, 3H). 13C
NMR (100 MHz, CDCl3): δ 206.6, 166.7, 148.0, 135.0,
131.3, 129.0, 128.5, 126.8, 54.0, 50.0, 49.3, 45.3, 43.4,
37.9, 37.1, 36.3, 35.7, 35.4, 34.2, 31.9, 28.8, 28.3, 20.9,
17.7, 13.2, 12.3. ESI-MS m/z: 418 [M-H]−.
RESULTS AND DISCUSSION
The synthetic steps are outlined in Figure 1.
Firstly, The C-C double bond was formed by Wittig
reaction with the Z-configuration [Deng et al., 1999].
Then, the C-N single bond at C-3 position was intro-
duced by a Mitsunobu reaction. To induce the C-3
amino group, a trial-and-error approach is required,
e.g., introduction of C-3 nitro group, C-3 azides group
[Czako et al., 2009], or C-3 phthalimide group
[Hamilton et al., 2012]. Only the C-3 phthalimide
steroid was obtained successfully from C-3 hydroxyl
steroid by a Mitsunobu reaction in a 66.3% yield. After
hydrazinolysis, the C-3 amino steroids were obtained in
above 90% yield. Meanwhile, the allylic position of
(17E )-3α-Benzamidopregn-17(20)-en-16-one (20f)
1
Yield: 59.7%. H NMR (400 MHz, CDCl3): δ
7.80–7.78 (m, 2H), 7.52–7.44 (m, 3H), 6.49 (q,
J = 7.5 Hz, 1H), 6.34 (d, J = 6.4 Hz, 1H), 4.35 (br s,
1H), 1.85 (d, J = 7.5 Hz, 3H), 1.03 (s, 3H), 0.90 (s, 3H).
13C NMR (100 MHz, CDCl3): δ 206.4, 166.7, 147.9,
135.2, 131.3, 129.1, 128.6, 126.9, 54.3, 50.1, 45.2, 43.4,
41.2, 37.9, 36.3, 34.1, 33.1, 32.8, 31.8, 28.2, 26.0, 20.6,
17.7, 13.2, 11.5. ESI-MS m/z: 418 [M-H]−.
Cell Viability Assay
Cell viability assay was performed as described C-17 (20) double bond could be oxidized to obtain the
previously [Wu et al., 2010].
C-16 hydroxyl or carbonyl group. Due to the limitation
Drug Dev. Res.

86
WANG ET AL.
TABLE 1. Inhibition of Cancer Cell Invasion by E-Salignone Amide
Derivatives
a
a
a
No.
IC50 (μM)
No.
IC50 (μM)
No.
IC50 (μM)
7a
7b
7c
7d
27.54 0.35 13a 39.48 0.04 19a 16.12 0.44
10.63 0.43 13b 36.11 0.39 19b 0.24 0.04
19c 25.24 0.27
39.17 0.08 13c
1.63 0.16 13d
>50
6.95 0.11 19d
0.91 0.12
0.62 0.09
7e
2.48 0.31 13e 25.61 0.33 19e
7f
7g
8a
8b
8c
8d
27.31 0.05 13f 37.85 1.20 19f 15.13 0.13
24.38 0.56 13g 46.54 0.05 15 4.01 0.05
31.22 1.13 14a >50 20a 24.24 0.47
21.12 0.06 14b 41.63 0.65 20b 3.46 0.07
44.34 0.37 14c >50 20c 33.02 0.06
19.15 0.51 14d 42.73 0.08 20d 9.39 0.14
Fig. 3. The effects of compound 19b and LY294002 (positive control)
on the migration of MDA-MB -231 cells. Quantitative assessment of
the migrated cells to the wound area was expressed as IC50 values with
the mean SD of three independent experiments.
8e
8f
8g
23.76 0.47 14e 36.57 0.36 20e 6.543 1.12
31.91 0.14 14f 48.26 0.23 20f 23.07 0.53
22.44 0.61 14g
>50
16
9.61 0.51
LY294002
0.38 0.06
aIC50 values are the mean of three independent experiments; standard
deviation is given.
tives. Thus, compound 19b is 15-fold more potent than
that of 20b, while compound 19e is 10-fold more
of hydrazinolysis with ketone, the allylic position had to potent than 20e. On the other hand, the C-16 carbonyl
be oxidized to a carbonyl group indirectly, while pro- derivatives had better activities than other two types of
tecting the amino group to avoid oxidation. Finally, the derivatives, including compounds 7a–g, 8a–g, 13a–g,
synthesis of amide derivatives was accomplished by the and 14a–g. However, an accurate SAR could not be
reaction of various acids or acid chlorides with corre- established due to the insufficient number of com-
sponding C-3 amino steroids, including compounds 5, pounds. Compound 19b was also evaluated in wound
6, 11, 12, 17, and 18.
healing assay (Fig. 3) where it was more potent
Synthetic small molecules and NPs are potent (IC50 = 0.44 μM) than the positive control, LY294002
antimetastatic agents, acting via complex pathways that (IC50 = 1.25 μM).
involve inhibition of cell proliferation and blockade of
Other natural steroids and synthetic steroid
cell migration [Molinski et al., 2009; Scott et al., 2009; derivatives also inhibit invasion or migration in tumor
Oskarsson et al., 2010]. Accordingly, in order to achieve cells. For example, α-Chaconine isolated from Solanum
an understanding of the mechanism responsible for tuberosum inhibited metastatic A549 cell invasion/
antimetastatic actions, cytotoxic effects of small mol- migration in wound healing and Boyden chamber assays
ecules must be deemphasized while maintaining their and had antiproliferative and proapoptotic effects on
ability to inhibit cancer cell migration. The in vitro the growth of cancer cells originating from human skin,
cytotoxic activities of the novel E-salignone amide liver, prostate, breast, and colon [Shih et al., 2007].
derivatives against breast adenocarcinoma MDA-MB- Another steroid derivative, PG545, was evaluated in a
231 were evaluated using MTT assays. The results (data phase I clinical trial in cancer patients with more potent
not shown) show that the majority of the compounds do antimetastatic activities in both HUVECs (human
not display positive effects on the inhibition of cancer umbilical vein endothelial cells) and in vivo models
cell proliferation below a concentration of 70 μM.
[Ferro et al., 2012]. Dehydroepiandrosterone (DHEA),
An important characteristic of tumor metastasis is the most abundant adrenal steroid in humans resulted
the invasive and migratory ability of tumor cells. The in an inhibition of around 30% in cell migration in
synthesized amide derivatives were evaluated in the MDA-MB-231 cells at a concentration of 100 μM at
transwell invasion assay, where they inhibited MDA- 24 h [Lopez-Marure et al., 2011]. At the same concen-
MB-231 cell invasion with IC50 values in the range tration, it decreased the number of cells migrating into
0.24–50 μM (Table 1). Compound 19b exhibited the the wound area in MDA-MB-231 cells.
most potent inhibitory effect with an IC50 value of
In the present study, compound 19b, a steroid
0.24 μM compared with the positive control, LY294002 derived from E-salignone, was effective at lower con-
(IC50 = 0.38 μM). The structure–activity relationships centrations than DHEA, E-salignone, and LY294002
(SAR) were also studied. It is notable that the in transwell and wound-healing assays in MDA-MB-
3β-substituted steroid derivatives exhibited a better 231 cells. These results suggest that E-salignone amide
anti-invasion activities than the 3α-substituted deriva- derivatives, like compound 19b, may be promising lead
Drug Dev. Res.

ANTIMETASTATIC E-SALIGNONE AMIDE DERIVATIVES
87
Kong D, Yamori T. 2009. Advances in development of
phosphatidylinositol 3-kinase inhibitors. Curr Med Chem
16:2839–2854.
candidates for the development of new antimetastasis
agents for the treatment of human breast cancer.
Kong DX, Yamori T. 2010. ZSTK474, a novel phosphatidylinositol
3-kinase inhibitor identified using the JFCR39 drug discovery
system. Acta Pharmacol Sin 31:1189–1197.
ACKNOWLEDGMENTS
Lopez-Marure R, Contreras PG, Dillon JS. 2011. Effects of
dehydroepiandrosterone on proliferation, migration, and death of
breast cancer cells. Eur J Pharmacol 660 (2–3):268–274.
Mego M, Mani SA, Cristofanilli M. 2010. Molecular mechanisms of
metastasis in breast cancer—clinical applications. Nat Rev Clin
Oncol 7:693–701.
This work was supported by grants from the
National Natural Science Foundation of China
(NSFC) (81072540), and Tianjin Research Program
of Applied Basic and Cutting-edge Technologies
(11JCZDJC20900, 12JCZDJC25800).
Molinski TF, Dalisay DS, Lievens SL, Saludes JP. 2009. Drug devel-
opment from marine natural products. Nat Rev Drug Discov
8:69–85.
Oskarsson T, Nagorny P, Krauss IJ, Perez L, Mandal M, Yang G,
Ouerfelli O, Xiao D, Moore MA, Massague J, et al. 2010. Diverted
total synthesis leads to the generation of promising cell-migration
inhibitors for treatment of tumor metastasis: in vivo and mecha-
nistic studies on the migrastatin core ether analog. J Am Chem Soc
132:3224–3228.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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